The long term objectives of this proposal are to understand mechanisms of immunity against lipopolysaccharide (LPS)-smooth strains of P. aeruginosa and develop effective immunotherapies that would augment the treatment of infections. P. aeruginosa is a leading nosocomial pathogen and LPS- smooth strains are the initial colonizers among patients with cystic fibrosis, an additional potential target for vaccine intervention. This proposal seeks to provide a better understanding of the diversity of protective epitopes found on the O-side chains of clinically important strains in order to formulate a comprehensive vaccine. The vaccine candidates are called high molecular weight polysaccharides and represent large, hence immunogenic, forms of the O-side chain structures. Although a limited number of serogroups of P. aeruginosa (7-10) have been implicated as pathogens, within some of these serogroups there are slight variations in the basic oligosacchaaride repeat unit that gives rise to antigenic variants that are the basis for expression of subtype epitopes. The need to include representative subtype epitopes in a comprehensive vaccine is undefined. The first aim of this proposal will look at the role of serogroup and subtype-specific epitopes in protective immunity. These studies will include: an evaluation of the immunogenicity of serogroup and subtype epitopes expressed on different preparations of high molecular weight polysaccharide; evaluation of the protective capacity, in mice, of antibodies specific to serogroup and subtype determinants, and production of a multivalent vaccine designed to elicit immunity to relevant serogroup and subtype epitopes. The second aim will investigate the local and systemic correlates of protective immunity against P. aeruginosa colonization and infection using recombinant Salmonella strains, systemically administered polysaccharide vaccines, or passively delivered antibodies as inducers or effectors of immunity. The serum and mucosal antibody response to P. aeruginosa LPS will be measured following oral vaccination with recombinant Salmonella strains or systemically delivered high molecular weight polysaccharides. These vaccines will be used to compare oral and systemic immunization as a strategy to promote P. aeruginosa clearance from the GI tract, prevent dissemination from the GI tract during neutropenia, and prevent infections at a distant mucosal site, the eye. Confirmation of the role of antibody will be accomplished by producing isotype disparate, variable-region identical monoclonal IgM, IgG and IgA antibodies against P. aeruginosa LPS O-side chains that will be used in evaluating the role of different antibody isotypes using passive protection studies in the various animal models. Overall this work should provide the formula for a comprehensive P. aeruginosa O-antigen- specific vaccine, provide important information about protective epitopes on the bacterial surface and contribute important new data about the comparative efficacy of IgM, IgG and IgA antibodies in protection against mucosal colonization and systemic dissemination of P. aeruginosa.